Except for a few N and O group lineages restricted to Africa, most HIV-1 strains share their most recent common ancestor in group M.1 Different subtypes of group M show characteristic geographic patterns in their spread, with subtype C, for example, being a frequent HIV-1 subtype in India, China, Ethiopia, and the southern part of Africa, whereas subtype B has been shown to predominate in Europe, North America, and Australia.2 Subtype A viruses are most prevalent in central and eastern Africa and in eastern European countries constituting the former Soviet Union.3,4 In regions where 2 or more different strains are cocirculating, intersubtype recombination is common. Such recombinant forms can frequently occur in AIDS epidemics and are designated as circulating recombinant forms (CRFs) (for review, see articles by Kahn2 and Peeters5). To date, at least 15 different CRFs have been characterized (http://www.hiv.lanl.gov/content/hiv-db/CRFs/CRFs.html). It has been assumed that CRFs constitute 10% to 20% of newly identified strains; moreover, in some cases, recombination between CRFs has also been reported.6-8 In addition to circulating recombinants, the presence of multiple mosaic viruses that do not fulfill the requirements associated with CRFs has been shown recently; these are called unique recombinant forms (URFs). In some areas, URFs exhibit remarkable diversity with multiple recombination breakpoints.9
The spread of HIV-1 in eastern European countries has been closely linked with the rise of injecting drug use, which increased promptly after the collapse of Soviet Union in the 1990s.10 The HIV-1 epidemic among intravenous drug users (IDUs) started in 1996 in southern Ukraine with subtype A and, to a limited extent, subtype B.11,12 In Estonia, during the 1990s, the reported number of HIV-1 cases was relatively low and the predominant HIV-1 type was B (likewise in other Baltic countries), with a limited distribution of subtypes A, C, D, F, and G as well as CRF02_AG.13,14 For example, in 1999, Estonia reported 96 diagnosed HIV cases, only 4% of which were detected among IDUs. This situation changed drastically in August 2000 when the number of IDU-associated HIV-positive cases started to increase rapidly and comprised nearly 90% of the new HIV cases reported in 2000.15 Most new HIV cases are still associated with IDUs, although the role of heterosexual transmission has gradually risen from less than 10% in 2000 to approximately 33% by the end of 2003 (unpublished data, 2003, AIDS Prevention Center, Tallinn, Estonia). A recently published study has shown a role of recombinant strains for the Estonian epidemic.16
This study was initiated to establish the exact genetic structure of the HIV-1 strains causing the IDU-associated epidemic outbreak of HIV-1 infection in Estonia after August 2000.
MATERIAL AND METHODS
Selection and Preparation of Samples
One hundred forty-one blood samples were obtained from HIV-1-positive individuals. Eighty-seven percent (n = 123) of the samples came from persons who identify themselves as IDUs and were infected during the outbreak of the virus in 2000 through 2003. Six samples belong to long-term patients infected sexually in 1988 through 1994, and 12 samples are from patients who were infected sexually in 2000 through 2003. Sample collection roughly represents the present situation of HIV-1 distribution in Estonia geographically by age, by gender, and by route of transmission. All subjects provided informed consent when giving blood for testing.
The blood samples were collected in BD Vacutainer Cell Preparation Tubes (CPTs; BD Diagnostics, Becton Dickinson, Mountainview, CA), and peripheral blood mononuclear cells (PBMCs) were isolated as buffy coats by density gradient centrifugation. Subsequently, genomic DNA was extracted and purified from the samples using the Qiamp Blood Mini Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. For preparation of the viral genomic RNA, the material from 1 mL of patient plasma was extracted using Qiaquick Viral RNA-kits (QIAGEN) and eluted in 30 μL of sterile RNAse-free water. Two microliters of extracted RNA was translated into complementary DNA (cDNA) using Superscript RT (Stratagene).
Polymerase Chain Reaction and Sequencing
Two proviral genomic regions were selected to identify existing strains and to detect possible recombination events: a 430-base pair (bp) fragment of the gag/pol region (corresponding to 1915-2344 bp in the reference HIV-1 sequence HXB2) and a 387-bp fragment of gp41 (corresponding to 7880-8266 bp in HXB2). Both regions have been shown to contain nucleotide divergence that is sufficient for subtype determination; yet, both are conserved enough to permit the design of conserved primers and to create the DNA sequence alignments.16-20 Nested polymerase chain reaction (PCR) was performed from purified genomic DNA samples using primers bjgag1 (5′TAGAAGAAATGATGACAGCATG3′), bjpol2 (5′TGGCTTTAATTTTACTGGTACAAG3′), msgag7 (5′GATGACAGCATGTCAGGGAG3′), and bjpol3 (5′GTTGACAGGTGTAGGTCCTAC3′) for the gag/pol region, as previously described,16-18,20 and primers gp40F1 (5′TCTTAGGAGCAGCAGGAAGCAGTATGGG3′), gp41R1 (5′AACGACAAAGGTGAGTATCCCTGCCTAA3′), gp46F2 (5′ACAATTATTGTCTGGTATAGTGCAACAGCA3′), and gp47R2 (5′TTAAACCTATCAAGCCTCCTACTATCATTA3′) for the gp41 region, as described in the study by Pieniazek et al.19 Nested PCR was followed by sequencing using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Buckinghamshire, United Kingdom).
Amplification of near-full-length proviral genomic DNA was carried out from PBMC genomic DNA samples by nested PCR, resulting in a set of overlapping DNA fragments. The following primers were used: gag-region nef1 (5′CAAGGGACTTTCCGCTGGGGAC3′), jupp1f (5′CTCGAAAGCGAAAGTTCCAGAG3′), jupp1r (5′ATTTGCATAGCTGCCTGGTGTC3′), and gagakr (5′GTGTAGCTGCTGGTCCTAATGC3′); pol-region jupp3f (5′GGAGCAGATGATACAGTATTAGAAG3′), jupp3r (5′CCATGTATTGATAGATCACCATTTC3′), xfow (5′CTTCCACAGGGATGGAAAGG3′), and yrev (5′TGCTCCTACTATGGGCTCTG3′); accessory gene-region afow (5′CCCTAGTCTGGCAGACCAAC3′) and brev (5′GGGTACACAAGCATGTGTAG3′); env-region jupp8r (5′GTTAATAGTAGTCCTGTAATGTTTG3′), jupp9f (5′GAATAACATGACCTGGATAGAATG3′), and jupp9r (5′TGGTCTTAAAGGCACCTGAG3′); and nef-region nefy (5′ATGGGTGGCAAGTGGTCAAA3′) and nef1 (5′CAAGGGACTTTCCGCTGGGGAC3′). PCR products were cloned into pTZ57R/T or pUC18 vector and sequenced using M13F24 and M13R22 universal sequencing primers (MBI Fermentas, Vilnius, Lithuania). As a next step, the obtained fragments were assembled into contiguous sequences.
From reverse-transcribed viral genomic RNA samples, virtually the entire genome was further amplified by nested PCR into 4 segments using the following primers: gag-region f12 (5′AAATCTCTAGCAGTGGCGCCCGAACAG3′), f14 (5′TCTCTCGACGCAGGACTCGGCTTG3′), G40 (5′GACTAGCGGAGGCTAGAAG3′), BJGAG3 (5′TCCCTAAAAAATTAGCCTGTC3′), BJPOL2 (5′TGGCTTTAATTTTACTGGTACAG3′), and BJPOL3 (5′GTTGACAGGTGTAGGTCCTAC3′); pol-region MSGAG7 (5′GATGACAGCATGTCAGGGAG3′), Int3′-1 (5′CAATCATCACCTGCCATCTG3′), pol5′-2 (5′CCCCTAGRAAAAAGGGYTGT3′), and pol3′-11 (5′GGRTCTCTGCTGTCYCTGTAA3′); accessory gene-region vif-vpu5′out (5′ATCCCCAAAGYCAAGGAGTA3′), vif-vpu3′out (5′TCCAYACAGGYACCCCATA3′), vif-vpu5′in (5′GACAGCAGTACARATGGCAG3′), and vif-vpu3′in (5′CTCTCATTGCCACTRTCYTC3′); and env-region JL86 (5′TGCTGTTTATTCATTTCAGAATTGG3′), JL89 (5′TCCAGTCCCCCCTTTTCTTTTAAAAA3′), JL88 (5′TAAGTCATTGGTCTTAAAGGTACCTG3′), and ed3 (5′TTAGGCATCTCCTATGGCAGGAAGAAGCGG3′). PCR products were directly sequenced after Qiaquick PCR cleanup-kit (QIAGEN) purification and primer walking using Bigdye 3.0 fluorescent terminator sequencing chemistry (Applied Biosystems) and the inner PCR primers. Fragments were assembled into contiguous genome sequences.
Sequence data were aligned using BioEdit Sequence Alignment Editor,21 version 5.0.9, (T. Hall, 2001; distributed by the author) and Align Plus 5, version 5.01 (Scientific & Educational Software) software. The alignments were corrected manually and subjected to a basic local alignment search tool (BLAST) search against the HIV database (http://www.hiv.lanl.gov/content/hiv-db/BASIC_BLAST/basic_blast.html). Informative sites were determined, and phylogenetic analysis was carried out using a Kimura 2-parameter 22 model and neighbor-joining (NJ) approach23 with 1000 bootstrap replications in Molecular Evolutionary Genetics Analysis, version 2, software (MEGA2).24
For identification of intersubtype recombination breakpoints, a bootscanning analysis25 was carried out using SimPlot,26 version 2.5 (S. C. Ray, 1999; distributed by the author) and PHYLIP (Phylogeny Inference Package),27 version 3.5c (J. Felsenstein, 1993; distributed by the author) software.
As represented in Figure 1A, the phylogenetic tree based on the gp41 region grouped most (n = 131) of the Estonian isolates from recently infected individuals together in a single lineage cluster with low pairwise genetic nucleotide diversity (d = 0.003 ± 0.001), supported by a high bootstrap value (see group I in Fig. 1A). A BLAST search of the sequences of this cluster gave highest similarity with CRF06_cpx type HIV-1 sequences. Consistent with this, 4 known CRF06_cpx sequences and several subtype G sequences (the CRF06_cpx shows similarity to G-type sequences in the gp41 region selected) were closely related to the Estonian isolates.
The remaining sequences from recent infections (n = 4) clustered with subtype A sequences (see group II in Fig. 1A). All Estonian subtype A-like sequences (d = 0.015 ± 0.005) demonstrated remarkable similarity with a subtype A sequence isolate (98UA0116) derived from Ukraine. Closely related variants of this isolate and recombinants derived from it are thought to be responsible for the explosive epidemic among a large number of Ukrainian IDUs since 1996.3,12,28,29 As expected, the samples of long-term patients (n = 6) grouped together with subtype B reference sequences (d = 0.047 ± 0.007; see group III in Fig. 1A).
The NJ tree constructed from the gag/pol sequences revealed the existence of 3 distinct clades with substantial bootstrap support among Estonian samples (see Fig. 1B). Most (93%) of the gag/pol lineages again showed the highest similarity with the CRF06_cpx and subtype G sequences (CRF06_cpx also has a subtype G-like sequence in the gag/pol region), whereas the remaining few sequences clustered together with subtype A and B reference sequences. Two distinct groups of isolates could be distinguished on the tree within the major CRF06_cpx clade (see groups IA and IB in Fig. 1B). The first group, including most of the sequences (n = 108), was composed of highly homogeneous lineages with an average pairwise genetic distance (d = 0.008 ± 0.001; see group IA in Fig. 1B). In 8 sequences showing long branch lengths (083, K19, 041, K5, K35, TV44, TV5, and TV77) a G-to-A hypermutation pattern was observed.
The second group in CRF06_cpx clade (see group IB in Fig. 1B) comprises a diverse set of sequences (d = 0.019 ± 0.004). An initial BLAST search and alignment of these sequences exposed their mosaic structure in the gag/pol region resembling subtype A in the first part of the segment and CRF06_cpx in the second. For a more detailed analysis, 2 consensus sequences were generated: the first from the Estonian homogeneous cluster of CRF06_cpx-like sequences (see group IA in Fig. 1B) and the second from the Estonian subtype A-like sequences (see group II in Fig. 1B). Comparison of the mosaic gag/pol sequences at the positions where the 2 consensus sequences differed revealed the presence of multiple different types of mosaic forms, with each type showing a different pattern of potential crossover points. Sequences that differed from each other by less than 3 consecutive character changes were grouped together, defining 9 different groups (Fig. 2).
Four sequences in the gag/pol region and 4 sequences in the gp41 region of Estonian IDU samples showed high sequence homology with subtype A. Interestingly, among these, there were 3 samples that showed homology with subtype A in the gag/pol region clustered together with CRF06_cpx-type sequences in the gp41 region, and vice versa. Only 1 isolate, EST2002-394, showed a subtype A sequence in both regions (see Fig. 1; Table 1).
The long-term sexually infected patients formed an independent group (d = 0.043 ± 0.005) intermingling with similar B lineages uncovered elsewhere in Europe (see group 3 in Fig. 1B). All sequences (n = 12) from sexually infected individuals who were infected with HIV after August 2000 showed the highest similarity to CRF06_cpx sequences in the gag/pol and gp41 regions.
To study the more prevalent Estonian HIV-1 forms in more detail, 2 representative HIV-1 near-full-length proviral genomes (EE0359 as a common variant among Estonian IDUs and EE0369 as a putative new recombinant form) were sequenced. Two additional nearly complete sequences were generated from viral genomic RNA by reverse transcriptase PCR from samples EST2002-1169 (another putative recombinant form) and EST2002-394 (subtype A according to gag/pol and gp41 sequences).
An NJ tree of these sequences (samples EE0359, EE0369, and EST2002-1169), together with CRF06_cpx reference genomes, is shown in Figure 3. In contrast to the other sequences, the EST2002-394 isolate clustered with eastern European subtype A sequences. The results of the bootscanning analyses confirmed that 2 of the 4 sequences (isolates EE0369 and EST2002-1169) were recombinants between subtype A and CRF06_cpx. They both share a similar structure, CRF06_cpx/A/CRF06_cpx (Fig. 4), belonging to recombinant form type 4 (see Fig. 2). When subjected to the BLAST search, the approximately 900-bp fraction of the subtype A-like sequence showed remarkable similarity to several subtype A and CRF03_AB forms of HIV-1 isolated from Ukraine, Russia, and Belarus.
As previously has been reported, this study confirms that a rare CRF of HIV-1, CRF06_cpx, is predominant in the population of Estonian HIV-1-positive individuals.16 CRF06_cpx is a complex mosaic form of the virus composed of successive fragments of subtypes A, G, K, and J and shows similarity to G sequences in the selected gp41 and gag/pol regions.30,31 Four near-full-length genomes of CRF06_cpx have been sequenced previously.30,32,33 All originated in West Africa, whereas sequencing of shorter fragments has reveled that this virus variant has also been introduced to other continents (at least to Europe and Australia). Although CRF06_cpx is spreading in different countries, its prevalence has been documented to be low.5,31,32
Studies carried out in the 1990s determined subtype B to be a predominant form of HIV-1 in the Estonian population.13,14 In accordance with the NJ tree based on the gp41 or gag/pol region, individuals with a long history of infection formed an independent group intermingling with similar B lineages uncovered elsewhere in Europe (see group III in Fig. 1A, B). This finding, as well as the highest genetic diversity of this clade in Estonians, possibly suggests that the B lineages in Estonia do not derive from a single recent founder-a conclusion consistent with this population's medical histories tracing back 10 years or longer.
The data presented here, and in an earlier study, demonstrate that the burst of the IDU-associated epidemic outbreak of HIV-1 infection since 2000 coincides with the drastic shift in genetic structure of HIV-1 in Estonia.16 The phenomenon that the beginning of a new epidemic or change in the epidemic pattern is frequently accompanied by a change in the molecular structure of the virus has been observed in Thailand, Uruguay, Argentina, and South Africa, for example.34-36 In Estonia, the new epidemic is among IDUs as opposed to sexually exposed people. The CRF06_cpx-like IDU virus isolates that are circulating in Estonia form a remarkably homogeneous cluster with an average pairwise genetic distance (d) of 0.003 ± 0.001 in the gp41 region, which is more than 10-fold lower than the average distance between Estonian B isolates (0.047 ± 0.007). This finding also suggests a recent introduction of the CRF06_cpx strain to Estonia, probably through a single carrier. Because of the small number of available reference sequences from geographically remote areas, the significant difference of our sequences from other CRF06_cpx isolates comes as no surprise. Hence, the origin of the CRF06_cpx lineage in Estonia remains unknown until other isolates with higher sequence similarity are found. The remarkably high incidence of a single particular lineage with low genetic variability in Estonia is consistent with the country's small area, population size, and transmission type.
A high incidence of different recombinant forms in the Estonian IDU population indicates that coinfection with different strains of HIV-1 occurs frequently among drug users. The fact that 1 particular type (type 4, see Fig. 2) comprises one third of all unique recombinant forms in Estonia, supported by data on 2 full-length and 6 partial sequences with the same recombinant structure, allows us to designate this as the emergence of a new CRF. It is noteworthy that the crossover sites of Estonian gag/pol URFs correspond to the genomic region (5′ end of pol) of HIV-1, which has been characterized as a recombination “hot spot”.37,38 As summarized in Table 1, in many individuals, discordant results of subtype determination became obvious between gag/pol and gp41 sequence data. In principle, this finding could reflect the existence of additional recombinant forms or the presence of 2 circulating strains in a single individual. Although a crossover site (gag/pol CRF06_cpx/A, gp41 CRF06_cpx) was detected inside sequenced regions for 23 samples, the presence and exact location of the recombination spot in 4 samples with discordant phylogenetic affiliations in the gp41 and gag/pol regions (see Table 1) remained outside the sequenced regions.
To conclude, first, we have confirmed the predominance of a rare HIV-1 form, CRF06_cpx, in the Estonian HIV-1-infected population. This is in strong association with the rapid spread of intravenous drug use. Second, we report the first complete CRF_06cpx-type provirus sequence in Europe. More data from Eastern Europe are needed to trace the origin and trafficking of the unusual HIV-1 subtype. Finally, a high proportion of the Estonian variants in gag/pol sequences were shown to fall into multiple types of CRF06/subtype A mosaics. A high frequency of some of the newly described unique recombinant types suggests that they may contribute to the pool of circulating genetic forms of HIV-1 in Estonia and neighboring countries.
The authors thank Jüri Parik for his advice and helpful discussions throughout the course of this study and Jaan Lind for excellent technical assistance. We are grateful to Katrin Kaldma, Tiit Talpsep, Olev Lumiste, and Tartu University Hospital Blood Center personnel for their kind help. The authors thank Michael Tristem for support in preparing the manuscript. This work was partially funded by basic research grant 0182566503 from the Estonian government.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
circulating recombinant form CRF06_cpx; intravenous drug users; unique recombinant forms